Robust antiviral activity of commonly prescribed antidepressants against emerging coronaviruses: in vitro and in silico drug repurposing studies

During the current coronavirus disease 2019 (COVID-19) pandemic, symptoms of depression are commonly documented among both symptomatic and asymptomatic quarantined COVID-19 patients. Despite that many of the FDA-approved drugs have been showed anti-SARS-CoV-2 activity in vitro and remarkable efficacy against COVID-19 in clinical trials, no pharmaceutical products have yet been declared to be fully effective for treating COVID-19. Antidepressants comprise five major drug classes for the treatment of depression, neuralgia, migraine prophylaxis, and eating disorders which are frequently reported symptoms in COVID-19 patients. Herein, the efficacy of eight frequently prescribed FDA-approved antidepressants on the inhibition of both SARS-CoV-2 and MERS-CoV was assessed. Additionally, the in vitro anti-SARS-CoV-2 and anti-MERS-CoV activities were evaluated. Furthermore, molecular docking studies have been performed for these drugs against the spike (S) and main protease (Mpro) pockets of both SARS-CoV-2 and MERS-CoV. Results showed that Amitriptyline, Imipramine, Paroxetine, and Sertraline had potential anti-viral activities. Our findings suggested that the aforementioned drugs deserve more in vitro and in vivo studies targeting COVID-19 especially for those patients suffering from depression.

The hCoV-19/Egypt/NRC-3/2020 SARS-CoV-2 "NRC-03-nhCoV" virus 20 and the MERS-CoV isolate NRCE-HKU270 21 were propagated in Vero-E6 cells, as described previously with minor modifications 8,22 . Briefly, confluent VERO-E6 cell monolayers were infected with NRC-03-nhCoV or NRCE-HKU270 in infection medium (DMEM with 1% pen/strep, 0.2% Bovine Serum Albumin (BSA), and 1 µg/ml TPCK-treated trypsin) at a multiplicity of infection (MOI) of 0.1 for 2 h. Following virus adsorption, the infection medium containing virus inoculum was discarded and exchanged with new infection media. The infected VERO-E6 monolayers were incubated for 4 days post-infection at 37 °C in a humidified incubator and 5% CO 2 . The cell supernatants were harvested and centrifuged at 2500 rpm for at least 5 min to remove cellular remains. The clear supernatant containing propagated viruses were then aliquoted, preserved at − 80 °C freezer. An aliquot of the saved NRC-03-nhCoV or NRCE-HKU270 viruses were subjected to virus titration using plaque titration assay, as described previously 8,23 . www.nature.com/scientificreports/ The tested antidepressant FDA-approved drugs; Citalopram, Escitalopram, Paroxetine, Sertraline, Mirtazapine, Amitriptyline, Imipramine, and Eszopicolone; were kindly provided by the Egyptian Holding Company for Pharmaceuticals and Chemicals, and the Egyptian National Organization for Drug Control and Research in Egypt. 50 ) determination. To assess CC 50 of the selected antidepressants, stock solutions were prepared by dissolving the selected drugs in 10% DMSO "in 1X DMEM" and serially diluted them with 1X DMEM to prepare the various working concentrations (Concentration range = 1-1000 µMol). The CC 50 of each compound was assayed in Vero-E6 cells by using crystal violet assay as previously described 24 . Briefly, 100 µl of the VERO-E6 cell suspension were distributed into 96-well plates (3*10 5 cells/mL). The seeded plates were then incubated at 37 °C in 5% humidified CO 2 incubator for 24 h. Cell monolayers were then co-incubated with different concentrations of each drug in triplicates at 37 °C in 5% humidified CO 2 incubator. Seventy two hours later, the media supernatants were discarded, the cell monolayers were washed once with 1X PBS and fixed with 10% formaldehyde for 1 h at room temperature (RT). The plates were further dried and stained at RT with 0.1% crystal violet for 20 min on a bench rocker. The monolayers are then washed, dried, and the crystal violet dye in each well was then dissolved with 200 µL methanol for 20 min on a bench rocker at RT. Eventually, the absorbance was measured at λmax 570 nm using the Anthos Zenyth 200rt plate reader (Anthos Labtec Instruments, Heerhugowaard, Netherlands). The cytotoxicity of various concentrations compared to the untreated cells was determined using nonlinear regression analysis by plotting log inhibitor versus normalized response. 50 ). The IC 50 values for the tested compound were determined as previously described 8 , with minor modifications. Briefly, the VERO-E6 monolayers in 96-well tissue culture plates were then washed once with 1 × PBS. The NRC-03-nhCoV" virus "TCID 50 = 100) was co-incubated with serial diluted working concentrations of the tested drugs at 37 °C for 1 h. The Vero-E6 cells were treated with virus/drug mixtures and kept at 37 °C for 1 h. Untreated/infected cells represented the virus control, however untreated/ uninfected cells referred to the cell control. After 72 h of co-incubation at 37 °C in 5% CO 2 incubator, the cell monolayers were fixed with 100 μL of 10% formaldehyde for 20 min and stained with 0.1% crystal violet "in distilled water" for 15 min at RT. To dissolve crystal violet dye, 100 μL of the absolute methanol were added per well and the optical density of the color is eventually measured at 570 nm using the Anthos Zenyth 200rt plate reader (Anthos Labtec Instruments, Heerhugowaard, Netherlands). The IC 50 values were calculated using nonlinear regression analysis by plotting log inhibitor versus normalized response.

Inhibitory concentration 50 (IC
Quantitative real time RT-PCR assessment mRNA expression. Vero-E6 cell suspensions were cultured in 12-well tissue culture plates and incubated overnight to get confluent monolayers. The cells were washed two times with 1 × PBS and 100 µL of virus HCoV-19/Egypt/NRC-1/2020 with dilution of MOI = 0.05 was preincubated for 1 h at 37 °C with 5% CO 2 before being added to the cells with 100 µL of predefined non-cytotoxic concentrations of the selected compounds. The compounds/virus mixtures were then added to the corresponding wells. Treated wells, cell control, and virus control are incubated at 37 °C under 5% CO 2 for 1 h with rocked every l5 min to ensure homogenous exposure of the cells to infection and avoid drying of cells. After 1 h, inoculum was removed and followed by addition of 1000 µL of infection medium, then infected cells were incubated at 37 °C with 5% CO 2 for 48 h. An aliquot of 300 µL was collected every 24 h from each dilution in duplicate for viral titration using qRT-PCR. A part of the sample underwent viral RNA extraction using the QIAamp Viral-RNA Kit (Qiagen, Germany). The qRT-PCR assay (targeting ORF1b-nsp14 gene) in the presence of specific primers and probes were performed using Verso 1-step qRT-PCR Kit (Thermo, USA). The primer and probe sequences for the ORF1b gene assay are: 5′-TGG GGY TTT ACR GGT AAC CT-3′ (Forward; Y = C/T, R = A/G), 5′-AAC RCG CTT AAC AAA GCA CTC-3′ (Reverse; R = A/G) and 5′-TAG TTG TGA TGC WAT CAT GAC TAG -3′ (Probe in 5′-FAM/ZEN/3′-IBFQ format; W = A/T). The reaction setup and the thermal cycling conditions were performed as described previously 25 .

Mechanism of action(s).
To investigate whether the tested drugs with low IC 50 and high selectivity indices can directly hit the viral particle "virucidal effect" and/or interfer with viral adsorption and/or viral replication during virus replication cycle, plaque infectivity reduction assay was performed as described previously 8 . Docking studies. The drugs were subjected to four molecular docking studies using MOE 2019.012 suite 26 to virtually examine their antiviral effects against both SARS-CoV-2 and MERS-CoV spike (S) and main protease (M pro ) proteins and to propose their mechanism of action. The co-crystallized inhibitors of M pro proteins were used as reference standards in each corresponding docking process in order to compare the binding scores and interactions of the tested antidepressant drugs.
Preparation of the tested FDA-approved antidepressant drugs (1)(2)(3)(4)(5)(6)(7)(8). Each drug was sketched using the MOE builder, adjusted for partial charges, 3D protonated, subjected to energy minimization process, and stored as (.moe) extension as described before [27][28][29] . Then, they were collected in four different databases, two of them containing only the prepared drugs (1-8) for both SARS-CoV-2 and MERS-CoV S proteins docking processes, the third one containing the prepared drugs together with the co-crystallized inhibitor (N3, 9) (1-9) for SARS-CoV-2 M pro docking, and the fourth one containing the prepared drugs together with the two co-crystallized www.nature.com/scientificreports/ inhibitors (K36 9 and B1S 10) (1-10) for MERS-CoV M pro docking. Each database was saved as an MDB file with (.mdb) extension to be used in the corresponding docking process.

Preparation of the SARS-CoV-2 and MERS-CoV S and M pro pockets.
The Protein Data Bank was used to extract the 3D X-ray structures of the S and M pro of SARS-CoV-2 (PDB codes: 6VW1 30 and 6LU7 31 , respectively) and MERS-CoV (PDB codes: 5YY5 32 and 5WKJ 33 , respectively). Furthermore, the downloaded protein structures were subjected to the detailed preparation steps described earlier [34][35][36] and including correction, 3D hydrogen addition, and energy minimization to be ready for docking steps [37][38][39] .
Docking processes of SARS-CoV-2 and MERS-CoV S and M pro pockets. The previously mentioned four databases were used for four different docking processes into the prepared SARS-CoV-2 and MERS-CoV S and M pro pockets, respectively. For each docking process, the corresponding database was introduced in place of the ligand in a general docking process. The dummy atoms were applied to select the docking site as the largest pocket for the prepared SARS-CoV-2 and MERS-CoV protein in each case. The program specifications were adjusted as described before in details [40][41][42] and the docking process was initiated for each case. The tested compounds that achieved the best binding scores and modes for each docking process were selected for further investigations [43][44][45] . Two program validation processes were carried out before the docking steps by redocking the co-crystallized inhibitors at their corresponding M pro binding pockets [46][47][48] . The valid performance was confirmed by obtaining low RMSD values (< 2) between the co-crystallized and redocked inhibitor molecules in each case [49][50][51][52][53][54][55] . Moreover, the superimposed docked structure with the crystal structure was presented for each validation process in the supplementary data (Fig. SI1).

Results
Cytotoxicity and antiviral activity of the tested antidepressants drugs. Half

Mode of action.
To determine the mechanism of action for the promising drugs towards tested viruses, it was necessary to examine the mode of action. Generally, candidate antiviral drugs can interfere with viral replication cycle by exerting direct virucidal effect or indirectly by blocking the viral adsorption into the host cell receptors or impairing intracellular viral replication 8 . Interestingly, Amitriptyline and Paroxitine had a combination of viral inhibitory effect on the tested SARS-CoV-2 with remarkable virucidal effect and effects on virus replication (Fig. 4A). Likewise, Imipramine and Paroxetine showed virucidal and replication potential combination mechanisms against MERS-CoV. On the other hand, Sertraline showed viral inhibitory effect against MERS-CoV at virucidal and adsorption mechanisms (Fig. 4B).
Docking studies. By visualizing the M pro binding pocket of SARS-CoV-2, it was found that it contains a co-crystallized inhibitor (N3), while that of MERS-CoV contains two co-crystallized inhibitors (K36 and K1S). All of them fitted inside the binding pocket of M pro in an asymmetric manner. Molecular docking studies were performed for the eight examined drugs against both the S and M pro pockets of SARS-CoV-2 and MERS-CoV in order to propose a suitable mechanism of action for their antiviral potentials.
The docking results on SARS-CoV-2 and MERS-CoV S and M pro active sites showing their scores and binding modes were recorded in the four docking processes and compared to that of the docked inhibitors in case of M pro docking in both viruses as depicted in Table 1. www.nature.com/scientificreports/ Observing the docking scores and interactions of the eight examined drugs indicates their expected binding affinities and therefore the intrinsic activities as well. We decided to further examine the most promising four antidepressant drugs in their biological activities (Amitriptyline, Imipramine, Paroxetine, and Sertraline as depicted in Tables 1, 2,  On the other hand, Imipramine binding scores were found to be − 6.00 and − 6.29 kcal/mol on the S and M pro pockets of MERS-CoV, respectively. It bound Gln471 amino acid with a H-bond inside the S pocket, and also bound Gly149 amino acid with a H-bond inside the M pro pocket of MERS-CoV.
Paroxetine 7 showed promising binding scores of − 6.62, − 6.03, − 6.79, − 6.99 kcal/mol on the S and M pro active sites of SARS-CoV-2 and MERS-CoV, respectively. It got stabilized inside both the S and M pro binding pockets of SARS-CoV-2 without any amino acid interactions indicating highly recommended affinity and stability as well. However, it formed one H-bond with Ser457 amino acid inside the S pocket, and one pi-H interaction with His166 amino acid inside the M pro pocket of MERS-CoV.
Finally, Sertraline gave binding scores of − 6.17 and − 5.99 kcal/mol on the S and M pro pockets of MERS-CoV, respectively. It formed one pi-H interaction with Gln471 amino acid inside the S pocket and showed no binding interactions inside the M pro pocket of MERS-CoV.
Notably, the docked co-crystallized inhibitor of SARS-CoV-2 M pro pocket (N3, 9) achieved a binding score of − 8.47 kcal/mol with the formation of three H-bonds with Gln192, Asn142, and Met49 amino acids,

Discussion
Several clinical trials are currently conducted on COVID-19 patients to explore various drugs which may target different features of the disease pathophysiology 57 . One of the most important features of this disease is the excessive inflammation that occurs post infection with SARS-CoV-2. The excessive release of cytokines which is known as "cytokine storm" represents a serious condition that is responsible for respiratory dysfunction, pulmonary fibrosis, and eventually organ failure 58 . In a consistent manner, depressive episodes are commonly reported to be associated with elevated central and peripheral blood levels of pro-inflammatory cytokines and receptors including TNF-α, IL-1β, IL-6, C-reactive protein (CRP), sTNFR1, sTNFR2, soluble IL-2 receptors (sIL-2R) 59,60 . It is recently recognized that COVID-19 is associated with neurological and cerebrovascular disorders. Moreover, approximately 50% of the patients experience post recovery psychiatric diseases 61 , in which the diseases' mechanisms are still unclear. However, latest reports suggested strong relationship between the inflammation involving the high cytokines levels and the psychiatric depressive disorders 62,63 . Consequently, the use of antidepressant drugs among the medications prescribed for COVID-19 patients is increasingly expected. This observation led to explore any possible antiviral activity of a bundle of commercially available antidepressants in the market. The list of tested antidepressentants were also selected to cover variable popularity indices (PI) in real clinical practice including amitriptyline (PI = 1.06), paroxetine (2.09), citalopram (PI = 1.47), escitalopram (PI = 0.83) and sertraline (1.66) 64 .We envisioned that if a certain antidepressant drug has a potential  www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ inhibitory effect on SARS-CoV-2, this will be a double weapon for conquering COVID-19 not only by relieving of the associated psychiatric disorders but also through inhibition of virus replication. Antidepressant drugs, particularly some SSRIs and TCAs exhibit anti-inflammatory activities and they can easily diffuse in the central nervous system 65 . A recent report suggested that many antidepressant drugs may potentially reduce the activity of acid that subsequently may hinder SARS-CoV-2 infection to the epithelial cells 66 . Additionally, a clinical study has been performed on hospitalized COVID-19 patients and the authors signified the relationship between the intake of antidepressants and decreased mortality risks. Carpinteiro et al., reported that amitriptyline, imipramine, fluoxetine, sertraline, escitalopram, or maprotiline's inhibition of acid sphingomyelin as well as genetic downregulation of it can prevent SARS-CoV-2 infection of either the cultured cells or the freshly separated human nasal epithelial cells 66 . In another clinical trial, treatment with SSRI fluvoxamine drug among high-risk outpatients with early diagnosed COVID-19 reduced significantly the hospitalization rate 67 . In line with previous studies, our in vitro and in silico studies demonstrated the potential ability of Amitriptyline and Paroxetine to effectively inhibit SARS-CoV-2. Also, Imipramine, Sertraline, and Paroxetine showed antiviral activity against MERS-CoV. The aforementioned docking discussions of the FDA-approved antidepressant drugs (amitriptyline, imipramine, paroxetine, and sertraline) against both SARS-CoV-2 and MERS-CoV pockets (S and M pro ) clarified greatly the binding affinities and therefore the expected intrinsic activities of the previously mentioned drugs towards the SARS-CoV-2 and MERS-CoV. Taking into consideration all this, besides the high safety records proved by the post marketing experience of antidepressant drugs, we encourage clinical trials for repositioning the use of these antidepressants to manage COVID-19 disease.

Conclusion
This study proposed the effective antidepressants drugs against SARS-CoV-2 (Amitriptyline and Paroxetine) and MERS-CoV (Imipramine, Sertraline, and Paroxetine), to be further evaluated by extra in vitro and in vivo experiments in order to obtain an effective antidepressant therapeutic targeting coronaviruses to be recommended to patients suffering from depression. Furthermore, these drugs could be used as lead compounds for further optimization to get more promising molecules with promising activities towards SARS-CoV-2.
One limitation of this study is that it did not cover all commercial antidepressants especially the novel unclassified antidepressants including ketamine, agomelatine and vortioxetine. Agomelatine is a novel antidepressant as well as a potent agonist of melatonin (MT), MT1 and MT2 receptor types and an antagonist of the serotonin (5HT) 68 . Vortioxetine is also a novel antidepressant capable of improving depressive and cognitive symptoms associated with major depressive disorder (MDD) 69 , while Ketamine is a rapid-acting and novel therapeutic treatment for treatment-resistant depression, which has also been demonstrated to attenuate symptoms of anhedonia 70 . Therefore we highly propose the testing of the in vitro antiviral activity of these novel antidepressants against COVID-19 in future studies.

Data availability
All data analysed during the current study are available from the corresponding author on reasonable request. Data that support the findings of this study are available within the paper and its supplementary data.